The role of cavities in protein dynamics: crystal structure of a photolytic intermediate of a mutant myoglobin

Abstract

We determined the structure of the photolytic intermediate of a sperm whale myoglobin (Mb) mutant called Mb-YQR [Leu-(B10)-->Tyr; His(E7)-->Gln; Thr(E10)-->Arg] to 1.4-A resolution by ultra-low temperature (20 K) x-ray diffraction. Starting with the CO complex, illumination leads to photolysis of the Fe-CO bond, and migration of the photolyzed carbon monoxide (CO*) to a niche in the protein 8.1 A from the heme iron; this cavity corresponds to that hosting an atom of Xe when the crystal is equilibrated with xenon gas at 7 atmospheres [Tilton, R. F., Jr., Kuntz, I. D. & Petsko, G. A. (1984) Biochemistry 23, 2849-2857]. The site occupied by CO* corresponds to that predicted by molecular dynamics simulations previously carried out to account for the NO geminate rebinding of Mb-YQR observed in laser photolysis experiments at room temperature. This secondary docking site differs from the primary docking site identified by previous crystallographic studies on the photolyzed intermediate of wild-type sperm whale Mb performed at cryogenic temperatures [Teng et al. (1994) Nat. Struct. Biol. 1, 701-705] and room temperature [Srajer et al. (1996) Science 274, 1726-1729]. Our experiment shows that the pathway of a small molecule in its trajectory through a protein may be modified by site-directed mutagenesis, and that migration within the protein matrix to the active site involves a limited number of pre-existing cavities identified in the interior space of the protein.

Figure 1

(A) Distal site pocket in Mb-YQR. The heme, its ligands (O₂ and CO), and residues Tyr(B10)29, Gln(E7)64, and His(F8)93 are shown in the following ligation states: deoxygenated, blue; oxygenated, red; carbonmonoxy, purple; CO* photolyzed at 20 K, green. The displacements of the hydroxyl group of Tyr(B10)29 and the amino group of Gln(E7)64, respectively, in going from the oxygenated to the carbonmonoxy state, are given in Å. It also may be noted that the side chain of Tyr(B10)29 slightly “bounces” toward the outside of the pocket in the photolyzed state with respect to the CO-bound state. (B) Electron density maps in the heme region calculated by using the measured structure factor amplitudes for Mb-YQR⋯CO* at 20 K minus the ones from Mb-YQR-CO bound. The map is contoured at 3-σ electron density. Shown are the bound CO of Mb-YQR-CO in a negative density region (red) and CO* clearly positioned in an elongated sphere of positive density (yellow). In addition, the position of other residues in the structure of the CO bound and the photolyzed states change: the iron moves out the porphyrin plane, and the proximal His(F8)93 (Lower) and Tyr(B10)29 (Upper) shift.

Figure 2

Stereo view of the docking site of the photolyzed CO* in Mb-YQR at 20 K. The site is defined as being formed by the residues having at least one atom at 5 Å or less from one of the two atoms of CO* and include: Gly(B6)25, Ile(B9)28, Tyr(B10)29, Gly(E8)65, Val(E11)68, Leu(E12)69, and Ile(G8)107. The distance of the CO* from the heme Fe is shown in Å.

Figure 3

The position of CO* in wt Mb and Mb-YQR, relative to residue at B10. Superposition of wt Mb⋯CO* (8) (dark) and [Mb-YQR⋯CO*] (light). For both proteins the model shows the heme, the photolyzed CO*, and the residue at position B10 i.e., Leu for wt Mb and Tyr for Mb-YQR. We also show: (i) the distance (2.9 Å) expected between the hydroxyl group of Tyr(B10)29 and the photolyzed CO* if this was to occupy the primary docking site (dark), characteristically observed in wt Mb; and (ii) the distance (2.2 Å) expected between Leu(B10)29 and the photolyzed CO* in the secondary docking site (light) identified in the photolyzed intermediate of Mb-YQR (see Fig. 1B).

Figure 4

Motions of Tyr(B10)29 in going from the photolyzed intermediate (where CO* is in the secondary docking site shown as 2 to the deoxygenated state. The position of CO* in the primary docking site is shown as 1. The figure depicts that approach of the ligand to the metal and rebinding would demand a substantial movement of Tyr(B10)29 from the unliganded configuration. Deoxy, light; photolyzed, dark.